Adenoviruses belong to the family Adenoviridae, which is divided into two genera, namely Mastadenovirus and Aviadenovirus (Horwitz, Shenk and Murphy, individually, In Virology, 3rd ed., Fields et al., eds., pages 2149-2171, 2111-2148, and 15-57, respectively (Raven Press, New York (1996)). Human adenoviruses are classified into six subgroups, namely A through F, based on hemagglutination patterns of erythrocytes from diverse animal species, and are further classified into over 49 serotypes (Horwitz et al., supra; and Schnurr et al., Intervirol., 36, 79-83 (1993)).
Adenoviruses are nonenveloped, regular icosahedrons of about 65 to 80 nm in diameter. The adenoviral capsid comprises 252 capsomeres, of which 240 are hexons and 12 are pentons. The hexons and pentons are derived from three different viral polypeptides (Maizel et al., Virology, 36, 115-125 (1968); Weber et al., Virology, 76, 709-724 (1977)). The hexon comprises three identical polypeptides of 967 amino acids each, namely polypeptide II (Roberts et al., Science, 232, 1148-1151 (1986)). The penton comprises a penton base, which provides a point of attachment to the capsid, and a trimeric fiber protein, which is noncovalently bound to and projects from the penton base.
The penton base, itself, is a ring-shaped complex comprising five identical proteinaceous subunits of polypeptide III (571 amino acids) (Boudin et al., Virology, 92, 125-138 (1979)). The penton base sequence is conserved among the various serotypes of adenovirus that have been sequenced (Neumann et al., Gene, 69, 153-157 (1988)).
The fiber protein comprises three identical proteinaceous subunits of polypeptide IV (582 amino acids) and comprises a tail, a shaft and a knob (Devaux et al., J. Molec. Biol., 215, 567-588 (1990)). The fiber shaft comprises repeats of 15 amino acids, which are believed to form two alternating .beta.-strands and .beta.-bends (Green et al., EMBO J., 2, 1357-1365 (1983)). The overall length of the fiber shaft and the number of 15 amino acid repeats varies between adenoviral serotypes. For example, the Ad2 fiber shaft is 37 nm long and comprises 22 repeats, whereas the Ad3 fiber is 11 nm long and comprises 6 repeats. Sequencing of over ten fiber proteins from different adenoviral serotypes has revealed a greater sequence diversity than that observed among other adenoviral proteins. For example, the knob regions of the fiber proteins from the closely related Ad2 and Ad5 serotypes are only 63% similar at the amino acid level (Chroboczek et al., Virology, 186, 280-285 (1992)), whereas their penton base sequences are 99% identical.
Ad2 and Ad5 fiber proteins, however, both likely bind to the same cellular receptor, since they cross-block each other's binding. In contrast, Ad2 and Ad3 fibers are only 20% identical (Signas et al., J. Virol., 53, 672-678 (1985)) and bind to different receptors (Defer et al., J. Virol., 64(8), 3661-3673 (1990)).
Other proteins, namely proteins IX, VI, and IIIa also are present in the adenoviral capsid. These proteins are believed to stabilize the viral capsid (Stewart et al., Cell, 67, 145-154 (1991); Stewart et al., EMBO J., 12(7), 2589-2599 (1993)).
An adenovirus, namely serotype 2 (Ad2), has been shown to use the fiber and the penton base to interact with distinct cellular receptors to attach to and efficiently infect a cell (Wickham et al., Cell, 73, 309-319 (1993)). First, the virus uses a receptor binding domain localized in the fiber knob (Henry et al., J. Virol., 68(8), 5239-5246 (1994)) to attach to an, as yet, unidentified cell-surface receptor (Phillipson et al., J. Virol., 2, 1064-1075 (1968); Wickham et al., Cell, 73, 309-319 (1993); Svensson et al., J. Virol., 38, 70-81 (1981); Hennache et al., Biochem. J., 166, 237-247 (1977); Defer et al. (1990), supra; and DiGuilmi et al., Virus Res., 38, 71-81 (1995)). Then, following viral attachment, the penton base binds to a specific member of a family of heterodimeric cell-surface receptors called integrins. For the Ad2 and Ad5 serotypes, which possess the long-shafted fibers, the penton base is not significantly involved in viral attachment to host cells (Wickham et al. (1993), supra).
The specificity with which an adenoviral penton base binds to an integrin is a function of the paired .alpha. and .beta. subunits of the integrin. For instance, the Ad2 penton base binds to integrins .alpha..sub.v .beta..sub.3 and .alpha..sub.v .beta..sub.5 (Wickham et al. (1993), supra; Nemerow et al., In Biology of Vitronectins and their Receptors, Preissner et al. (eds.), pages 177-184 (Elsevier Science Publishers (1993)); and Varga et al., J. Virol., 65, 6061-6070 (1991)). Some integrins, such as the .alpha..sub.v integrins, are present on the surface of nearly all cells, except for unstimulated hematopoietic cells (Gladson et al., In Integrins, Y. Takada (ed.), pages 83-99 (CRC Press, Boca Raton, Fla. (1994)), whereas other integrins have a narrower tissue distribution. In particular, .beta..sub.2 integrins are present only on leukocytes, such as neutrophils and macrophages, .alpha..sub.4 integrins are present only on lymphocytes and fibroblasts, and the .alpha..sub.IIb .beta..sub.3 integrin is present only on platelets and megakaryocytes. The integrin subunit complement of a cell, therefore, in some sense limits the infectability of that cell by different serotypes of adenovirus.
Most integrins recognize short linear stretches of amino acids in a ligand, such as the tripeptide RGD (i.e., Arg Gly Asp), which is found in the majority of extracellular matrix ligands. The integrin .alpha..sub.IIb .beta..sub.3 binds fibrinogen via the amino acid sequence KQAGD (i.e., Lys Gln Ala Gly Asp; SEQ ID NO: 1) (Kloczewiak et al., Biochemistry, 23, 1767-1774 (1984)), and .alpha..sub.4 .beta..sub.1 binds fibronectin via the core sequence EILDV (i.e., Glu Ile Leu Asp Val; SEQ ID NO: 2) (Komoriya et al., J. Biol. Chem., 266, 15075-15079 (1991)). Another structural motif, NPXY (i.e., Asn Pro Xaa Tyr; SEQ ID NO: 3), which is present in the .beta. subunits of .alpha..sub.v -containing integrins, also has been shown to be important for integrin-mediated internalization (Suzuki et al., PNAS (USA), 87, 5354 (1990)).
It appears that the RGD tripeptide also functions in the interaction of adenoviral penton base with .alpha..sub.v integrins. Exogenously added RGD peptides block the penton base from binding to cells (Wickham et al. (1993), supra), and adenoviruses with point mutations in the RGD sequence of the penton base are restricted in their ability to infect cells (Bai et al., J. Virol., 67, 5198-5205 (1993)).
The RGD tripeptide sequence is present in the hypervariable regions of Ad2 and Ad5 (both are subgroup C) penton bases, which are identical in the region of the RGD tripeptide sequence. Secondary structural analysis of the hypervariable regions of the RGD-containing penton bases of Ad2, Ad5, and Adl2 (subgroup A) predicts that, in each case, the RGD motif is flanked by .alpha.-helices, which are believed to form the spikes seen in cryo-electron micrographic (cryo-EM) images of the Ad2 penton base (Stewart et al., EMBO J., 12(7), 2589-2599 (1993)). The RGD tripeptide also is present in the penton base of Ad3 (subgroup B, which strongly hemagglutinates rhesus erythrocytes but not those of rat) and Ad4 (subgroup E).
Once Ad2 or Ad5 attaches to a cell via its fiber, it undergoes receptor-mediated internalization into clathrin-coated endocytic vesicles by penton base binding to integrins. The Ad2 or Ad5 (subgroup C) penton base is not significantly involved in viral attachment to the host cell (Wickham et al. (1993), supra), presumably due to the length of their fiber shafts (37 nm), which may sterically block a primary attachment event to .alpha..sub.v integrins. Ultimately, the viral particles are transported to the nuclear pore complex of the cell, where the viral genome enters the nucleus, thereby initiating infection.
The ability of adenovirus to enter cells efficiently has allowed the adenoviral-mediated targeted transfer of one or more recombinant genes to diseased cells or tissue in need of treatment. In fact, adenoviral vectors are preferred over other vectors commonly employed for gene therapy (e.g., retroviral vectors), since adenoviral vectors can be produced in high titers (i.e., up to about 10.sup.13 viral particles/ml) and they efficiently transfer genes to nonreplicating, as well as replicating, cells (see, for example, review by Crystal, Science, 270, 404-410 (1995)). Adenoviral vectors are especially preferred for somatic gene therapy of the lungs, given their normal tropism for the respiratory epithelium.
Other advantages that accompany the use of adenoviruses as vectors for gene therapy include: (1) the rare observance of recombination; (2) the absence of an ostensible correlation of any human malignancy with adenoviral infection, despite the common occurrence of infection; (3) the adenoviral genome (which is comprised of linear, double-stranded DNA) can be manipulated to carry up to about 7.5 kb of exogenous DNA, and longer DNA sequences can potentially be carried into a cell, for instance, by attachment to the adenoviral capsid (Curiel et al., Human Gene Therapy, 3, 147-154 (1992)); (4) an adenovirus is unlikely to interfere with normal cell function since the vector commands expression of its encoded sequences in an epichromosomal fashion; and (5) live adenovirus has been safely used as a human vaccine for many years.
A drawback to the use of adenovirus in gene therapy, however, is that all cells that comprise receptors for the adenoviral fiber and penton base will internalize the adenovirus, and, consequently, the gene(s) being administered--not just the cells in need of therapeutic treatment. Also, cells that lack either the fiber receptor or the penton base receptor, e.g., integrin, will be impaired in adenoviral mediated gene delivery (Silver et al., Virology, 165, 377-387 (1988); Horvath et al., J. Virol., 62, 341-345 (1988); and Huang et al., J. Virol., 69, 2257-2263 (1995)). Similarly, other cells, which appear to lack an adenoviral fiber receptor, are transduced by adenovirus, if at all, with a very low efficiency (Curiel et al. (1992), supra; Cotten et al., PNAS (USA), 87, 4033-4037 (1990); Wattel et al., Leukemia, 10, l71-174 (1996)). Accordingly, limiting adenoviral entry to specific cells and/or expanding the repertoire of cells amenable to adenovirus-mediated gene therapy would constitute a substantial improvement over the current technology. Such truly "targeted" adenoviral gene delivery also could potentially reduce the amount of adenoviral vector that is necessary to obtain gene expression in the targeted cells and, thus, potentially reduce side effects and complications associated with an increased dose of adenovirus.
In efforts to achieve cell targeting, adenovirus has been employed essentially as an endosomolytic agent in the transfer into a cell of plasmid DNA, which contains a marker gene and is complexed and condensed with polylysine covalently linked to a cell-binding ligand, such as transferrin (Cotten et al., PNAS (USA), 89, 6094-6098 (1992); and Curiel et al., PNAS (USA), 88, 8850-8854 (1991)). It has been demonstrated that coupling of the transferrin-polylysine/DNA complex and adenovirus (e.g., by means of an adenovirus-directed antibody, with transglutaminase, or via a biotin/streptavidin bridge) substantially enhances gene transfer (Wagner et al., PNAS (USA), 89, 6099-6103 (1992)). However, these approaches are somewhat less than desirable in that they require the ligation of the ligand, such as transferrin, with polylysine, and the advance preparation of the transferrin-polylysine DNA complexes. Moreover, the complexes formed with adenovirus could be endocytosed by binding either to cellular adenoviral receptors or to transferrin receptors. Additionally, polylysine, by itself, is capable of binding to cells, thereby interfering with the specificity of this approach.
In order to circumvent such non-specific binding of the adenovirus, the adenoviral fiber has been modified, either by incorporation of sequences for a ligand to a cell surface receptor or sequences that allow binding to a bispecific antibody (i.e., a molecule with one end having specificity for the fiber, and the other end having specificity for a cell surface receptor) (PCT international patent application no. WO 95/26412 (the '412 application); Watkins et al., "Targeting Adenovirus-Mediated Gene Delivery with Recombinant Antibodies," Abst. No. 336). In both cases, the typical fiber/cell surface receptor interactions are abrogated, and the adenovirus is redirected to a new cell surface receptor by means of its fiber. Some downfalls associated with the approach of the '412 application, which calls for modification of the fiber, are that such fiber modifications can require the need for different cell lines (i.e., cell lines having the receptor for which the modified virus is now targeted) to propagate the virus, and/or a different means of cell delivery (e.g., liposome-mediated delivery) to introduce adenovirus intracellularly. Moreover, the approaches of Watkins et al. and the '412 application appear to be limited to the use of the adenoviral fiber in cell targeting.
Another approach to targeted gene delivery involves administering a targeting element coupled to a first molecule of a high affinity binding pair, wherein the targeting element is capable of specifically binding to a selected cell type (PCT international patent application no. WO 95/31566). Then, a gene delivery vehicle coupled to a second molecule of the high affinity binding pair is administered, wherein the second molecule is capable of specifically binding to the first molecule, such that the gene delivery vehicle is targeted to the selected cell type. The sequential administration of the various components is probably done to prevent agglomeration of the vector particles, e.g., in cases where the targeting element is multivalent for the domain that recognizes the vector, which would reduce transduction efficiency. However, such sequential administration is disadvantageous, since it allows for internalization of the targeting element before it can complex with the vector. Furthermore, internalization of the preadministered targeting element clears the receptor from the cell surface, thereby preventing efficient targeting of the complexed targeting element and vector, and also potentially leading to impairment of the cell processes controlled by the receptors. Moreover, such premature internalization would necessitate the use of relatively high levels of the targeting element.
The present invention seeks to overcome many of the problems of the aforesaid approaches to recombinant adenoviral gene therapy. Accordingly, it is an object of the present invention to provide a method of targeting attachment of an adenovirus to a cell for cell entry, as well as recombinant adenovirus, vectors and other constituents for carrying out the method. These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the following detailed description.